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Abstract:

A method of producing a restorative material used to restore a
tooth-deficient area in an oral cavity, the method comprising:
positioning, in a support carrier, a first cell mass formed from either
mesenchymal cells or epithelial cells and a second cell mass formed from
the other of the mesenchymal cells or epithelial cells, one of the
mesenchymal cells or epithelial cells being derived from a tooth germ and
the first and second cell masses being not mixed with each other but made
to closely contact each other; culturing the first and second cell masses
to form a reconstructed tooth germ or tooth; and confirming
directionality of the reconstructed tooth germ or tooth formed by the
culturing so as to enable the reconstructed tooth germ or tooth to be
embedded in the tooth-deficient area such that a tip of the tooth faces
an interior of the oral cavity, the tooth germ or tooth whose
directionality has been confirmed being used as a restorative material to
obtain an equivalent of a missing tooth in the tooth-deficient area. A
method for restoring a tooth-deficient area, the method comprising;
embedding the reconstructed tooth germ or tooth obtained by the
production method in the tooth-deficient area.

Claims:

1. A method of producing a restorative material used to restore a
tooth-deficient area in an oral cavity, the method comprising:
positioning, in a support carrier, a first cell mass formed from either
mesenchymal cells or epithelial cells and a second cell mass formed from
the other of the mesenchymal cells or epithelial cells, one of the
mesenchymal cells or epithelial cells being derived from a tooth germ and
the first and second cell masses being not mixed with each other but made
to closely contact each other; culturing the first and second cell masses
to form a reconstructed tooth germ or tooth; and confirming
directionality of the reconstructed tooth germ or tooth formed by the
culturing so as to enable the reconstructed tooth germ or tooth to be
embedded in the tooth-deficient area such that a tip of the tooth faces
an interior of the oral cavity, the tooth germ or tooth whose
directionality has been confirmed being used as a restorative material to
obtain an equivalent of a missing tooth in the tooth-deficient area.

2. The method of producing a restorative material according to claim 1,
wherein the equivalent of a missing tooth has a Knoop hardness of enamel
of 300 to 600 KHN and a Knoop hardness of dentin of 60 to 120 KHN.

3. The method of producing a restorative material according to claim 1,
wherein the culturing is organ culturing and the restorative material
comprises a reconstructed tooth germ or tooth formed by the organ
culturing and a support carrier.

4. The method of producing a restorative material according to claim 1,
wherein both of the mesenchymal cells and the epithelial cells are
derived from a tooth germ.

5. The method of producing a restorative material according to claim 1,
wherein each of the first cell mass and the second cell mass consists of
a single cell.

7. A method of restoring a tooth-deficient area in an oral cavity, the
method comprising: positioning, in a support carrier, a first cell mass
formed from either mesenchymal cells or epithelial cells and a second
cell mass formed from the other of the mesenchymal cells or epithelial
cells, at least one of the mesenchymal cells or epithelial cells being
derived from a tooth germ and the first and second cell masses being not
mixed with each other but made to closely contact each other; culturing
the first and second cell masses to form a reconstructed tooth germ or
tooth; and embedding the reconstructed tooth germ or tooth in the
tooth-deficient area.

8. The method of restoring a tooth-deficient area according to claim 7,
wherein both of the mesenchymal cells and the epithelial cells are
derived from a tooth germ.

9. The method of restoring a tooth-deficient area according to claim 7,
wherein each of the first cell mass and the second cell mass consists of
a single cell.

Description:

TECHNICAL FIELD

[0001] The present invention relates to a method for restoring a
tooth-deficient area and a method for producing a restorative material.

BACKGROUND ART

[0002] A tooth is an organ having: enamel in the outermost layer and
dentin in its inner layer, both of which are hard tissues; odontoblasts,
which produce the dentin, inside the dentin; and dental pulp in the
central portion. Teeth may be lost by dental caries, periodontal diseases
or the like, and from the perspective of the significant influence of the
presence or absence of teeth on appearance and taste of food, and from
the perspective of maintaining health and a high quality of life, various
tooth regenerative techniques have been developed.

[0003] For example, J. Dent. Res., 2002, Vol. 81(10), pp. 695-700
discloses that, by intraperitoneally transplanting cells, such as
epithelial cells or mesenchymal cells isolated from a tooth germ, to a
mouse together with a biodegradative carrier, a tooth-like tissue is
regenerated.

[0004] For example, as a method for regenerating a tooth germ, Japanese
Patent Application Laid-Open (JP-A) No. 2004-331557 discloses a method in
which tooth germ cells isolated from a living body are cultured in the
presence of a physiologically active substance such as fibroblast growth
factor. Further, JP 2004-357567 A proposes a method in which at least one
type of cells selected from tooth germ cells isolated from a living body
and cells capable of differentiating thereinto are cultured together with
a carrier containing fibrin, wherein the carrier containing fibrin has a
shape that allows a tooth germ to be formed in a shape of interest,
thereby forming a "tooth" having a unique morphology.

[0005] WO 2006/129672 discloses a technique in which epithelial cells and
mesenchymal cells derived from a tooth germ are made into cell masses,
which are then placed in collagen gel such that these cell masses are
closely contacting with each other, and the cell masses are cultured
while being kept under such a condition, thereby producing a tooth having
a cell arrangement unique to a tooth.

[0006] On the other hand, WO 2003/101503 discloses a method in which tooth
germ cells and cells capable of differentiating thereinto are cultured
under mechanical stimulation to regenerate a tooth germ, and a
therapeutic method in which the thus-regenerated tooth germ is
transplanted to the jawbone of a patient wherein a tooth germ is lost or
damaged.

[0007] Further, JP 2005-013261 A discloses an artificial biomaterial
having colloidal silica particles attached on its surface as an implant
material to be used by being embedded in the living body.

[0008] However, it has been pointed out that implanting of a screw-shaped
implant made of a material such as titanium in implant therapy at present
suppresses growth of the jawbone during the growth period of jaw and
makes tooth migration impossible, which are problematic. Further,
although achievement of normal occlusion is necessary for a regenerated
tooth to have the same function as that of a normal tooth, such occlusion
has not been sufficiently confirmed in the above techniques using cells
isolated from the living body. Further, nerves in the periodontal
ligament have responsiveness to noxious stimuli of the tooth such as
compression, and this is biologically important in view of perception
such as feeling of food. For a regenerated tooth to have the same
function as that of a normal tooth, the tooth-deficient area is expected
to be restored such that the regenerated tooth allows normal occlusion to
have hardness equivalent to that of a normal tooth and the nerves extend
into the regenerated tooth whereby the tooth has normal responsiveness to
stimulation as a tooth.

DISCLOSURE OF THE INVENTION

Means for Solving the Problems

[0009] Accordingly, the object of the present invention is to provide: a
method for producing a restorative material to restore a tooth-deficient
area such that the regenerated tooth has hardness equivalent to that of a
normal tooth, such that normal occlusion is attained, and such that the
regenerated tooth has responsiveness to stimuli equivalent to that of a
normal tooth; and a method for restoring a tooth-deficient area.

[0010] The present invention provides a method for producing a restorative
material used for restoration of a tooth-deficient area in an oral
cavity, and a method for restoring a tooth-deficient area.

[0011] The first aspect of the present invention provides a method of
producing a restorative material used to restore a tooth-deficient area
in an oral cavity, the method including:

[0012] positioning, in a support carrier, a first cell mass formed from
either mesenchymal cells or epithelial cells and a second cell mass
formed from the other of the mesenchymal cells or epithelial cells, one
of the mesenchymal cells or epithelial cells being derived from a tooth
germ and the first and second cell masses being not mixed with each other
but made to closely contact each other;

[0013] culturing the first and second cell masses to form a reconstructed
tooth germ or tooth; and

[0014] confirming directionality of the reconstructed tooth germ or tooth
formed by the culturing so as to enable the reconstructed tooth germ or
tooth to be embedded such that the tip of the tooth faces the interior of
the oral cavity; and,

[0015] the tooth germ or tooth whose directionality has been confirmed
being used as a restorative material to obtain an equivalent of a missing
tooth in the tooth-deficient area.

[0016] The second aspect of the present invention provides a method of
restoring a tooth-deficient area in an oral cavity, the method including:

[0017] positioning, in a support carrier, a first cell mass formed from
either mesenchymal cells or epithelial cells and a second cell mass
formed from the other of the mesenchymal cells or epithelial cells, one
of the mesenchymal cells or epithelial cells being derived from a tooth
germ and the first and second cell masses being not mixed with each other
but made to closely contact each other;

[0018] culturing the first and second cell masses to form a reconstructed
tooth germ or tooth; and

[0019] embedding the reconstructed tooth germ or tooth in the
tooth-deficient area.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIG. 1A shows photographic images of the outer appearance of the
tooth taken at an angle of 45° with respect to the site of
transplantation in the maxilla of the tooth of Example 2 of the present
invention, which were taken during the eruption period (left), the period
after the eruption until occlusion (center) and the period after the
occlusion (right).

[0021]FIG. 1B shows photographic images of the outer appearance of the
tooth taken from the direction vertical to the site of transplantation in
the maxilla of the tooth of Example 2 of the present invention, which
were taken during the eruption period (left), the period after the
eruption until occlusion (center) and the period after the occlusion
(right).

[0022]FIG. 1C shows photographic images of occlusion of the tooth of
Example 2 of the present invention, which were taken during the eruption
period (left), the period after the eruption until occlusion (center) and
the period after the occlusion (right).

[0023] FIG. 1D shows CT images of the outer appearance of the tooth of
Example 2 of the present invention, which were taken during the eruption
period (left), the period after the eruption until occlusion (center) and
the period after the occlusion (right).

[0024] FIG. 1E shows cross-sectional CT images (E) of the tooth of Example
2 of the present invention, which were taken during the eruption period
(left), the period after the eruption until occlusion (center) and the
period after the occlusion (right).

[0025] FIG. 2A is a graph showing the Knoop hardness of enamel of the
erupted tooth of Example 2 of the present invention.

[0026] FIG. 2B is a graph showing the Knoop hardness of dentin of the
erupted tooth of Example 2 of the present invention.

[0027] FIG. 3 is a hematoxylin-eosin stained image of the regenerated
tooth of Example 2 of the present invention, which the tooth immediately
before its eruption (at a magnification of ×20).

[0028]FIG. 4A is a hematoxylin-eosin stained image to confirm the bone
remodeling phenomenon in the bone at the periodontal ligament (B:
pressure side, C: tension side) which occurred when the orthodontic force
of Example 2 of the present invention was loaded (at a magnification of
×20).

[0029]FIG. 4B is a magnified view showing the pressure side (black-framed
area B) in FIG. 4A (at a magnification of ×200).

[0030]FIG. 4C is a magnified view showing the tension side (black-framed
area C) in FIG. 4A (at a magnification of ×200).

[0031] FIG. 5A is an immunostained image, which shows expression of c-fos
in the medulla oblongata observed in the absence of the orthodontic
stimulations of Example 2 of the present invention (at a magnification of
×100).

[0032] FIG. 5B is an immunostained image, which shows expression of c-fos
in the medulla oblongata observed 2 hours after the application of
orthodontic stimulations of Example 2 of the present invention (at a
magnification of ×100).

[0033] FIG. 5C is an immunostained image, which shows expression of c-fos
in the medulla oblongata observed 48 hours after the application of
orthodontic stimulations (at a magnification of ×100).

[0034] FIG. 5D is a immunostained image, which shows expression of c-fos
in the medulla oblongata observed 2 hours after dental pulp exposure (at
a magnification of ×100).

BEST MODE FOR CARRYING OUT THE INVENTION

[0035] The method of the present invention of producing a restorative
material is a method of producing a restorative material used to restore
a tooth-deficient area in an oral cavity, the method including:

[0036] positioning, in a support carrier, a first cell mass formed from
either mesenchymal cells or epithelial cells and a second cell mass
formed from the other of the mesenchymal cells or epithelial cells, one
of the mesenchymal cells or epithelial cells being derived from a tooth
germ and the first and second cell masses being not mixed with each other
but made to closely contact each other;

[0037] culturing the first and second cell masses to form a reconstructed
tooth germ or tooth (this step is hereinafter referred to as a
"reconstructed tooth germ-formation step"); and

[0038] confirming directionality of the reconstructed tooth germ or tooth
formed by the culturing so as to enable the reconstructed tooth germ or
tooth to be embedded such that the tip of the tooth is faces the interior
of the oral cavity (this step is hereinafter referred to as a
"directionality-confirmation step");

[0039] the tooth germ or tooth whose directionality has been confirmed
being used as a restorative material to obtain an equivalent of a missing
tooth in the tooth-deficient area.

[0040] Further, the method of the present invention of restoring a
tooth-deficient area is a method of restoring a tooth-deficient area in
an oral cavity, the method including:

[0041] positioning, in a support carrier, a first cell mass formed from
either mesenchymal cells or epithelial cells and a second cell mass
formed from the other of the mesenchymal cells or epithelial cells, one
of the mesenchymal cells or epithelial cells being derived from a tooth
germ and the first and second cell masses being not mixed with each other
but made to closely contact each other;

[0042] culturing the first and second cell masses to form a reconstructed
tooth germ or tooth (this step is hereinafter referred to as a
"reconstructed tooth germ-formation step"); and

[0043] embedding the reconstructed tooth germ or tooth in the
tooth-deficient area (this step is hereinafter referred to as an
"embedding step").

[0044] In the method of the present invention for producing a restorative
material and the method of the present invention for restoring a
tooth-deficient area, the reconstructed tooth germ or tooth obtained in
the reconstructed tooth germ-formation step by positioning a first cell
mass and a second cell mass in a support carrier, which first and second
cell masses are not mixed with each other but made to closely contact
each other, is used as a restorative material to be embedded in the
missing area. By embedding this reconstructed tooth germ or tooth in the
missing area as a restorative material, an equivalent of a tooth erupts
from the embedding area that comes to have a length and hardness that
allow occlusion with the opposing tooth, and extension of nerves into the
equivalent may be achieved. Therefore, the equivalent of a tooth can have
hardness equivalent to a normal tooth similar to the teeth surrounding
the equivalent of the tooth, an ability to achieve normal occlusion, and
responsiveness to stimuli equivalent to a normal tooth.

[0045] In the present invention, the term "equivalent of a tooth" or the
like is used to mean a tooth that has a length and hardness that allow
occlusion with the opposing tooth when the tooth has erupted from the
tooth-deficient area where the equivalent was embedded.

[0046] In the present invention, the "reconstructed tooth germ-formation
step" is applied to both the method for producing a restorative material
and the method for restoring a tooth-deficient area

[0047] Further, the term "step" used herein includes not only a discrete
step, but also steps which cannot be clearly distinguished from another
step, as long as the expected effect of the pertinent step can be
achieved.

[0048] In addition, ranges indicated herein with "to" include the
numerical values before and after "to".

[0049] The invention will be described below.

[0050] In the present invention, the term "tooth" means a tissue
continuously having a layer of dentin inside and a layer of enamel
outside, a tissue having directionality resulting from a crown and a
root. The directionality of a tooth may be identified by arrangement of
its crown and root. The crown and root may be visually confirmed based on
their shapes, histological staining or the like. The crown means a part
having a layer structure of enamel and dentin, and the enamel layer is
absent in the root.

[0051] Dentin and enamel may be easily and morphologically identified by
those skilled in the art by histological staining or the like. Enamel may
also be identified by the presence of an ameloblast, which may be
confirmed by the presence/absence of amelogenin. On the other hand,
dentin may be identified by the presence of an odontoblast, which may be
confirmed by the presence/absence of dentinsialoprotein. Confirmation of
amelogenin and dentinsialoprotein may be carried out easily by a method
well-known in the art, and examples of the method include in situ
hybridization and immunostaining with an antibody, or the like.

[0052] Further, the directionality of a tooth may be identified based on
arrangement of its crown and root. The crown and root may be visually
confirmed based on their shapes, histological staining or the like.

[0053] In the present invention, the terms "tooth germ" and "tooth bud"
are used to refer specifically to those distinguished based on the
developmental stages. In this case, "tooth germ" means an early embryo of
a tooth, which is destined to become a tooth in the future, and which is
at a stage including the bud stage and the bell stage according to
typical developmental staging of a tooth, and especially means such a
tissue in which no accumulation of dentin and enamel is observed, which
are characteristic to the hard tissue of a tooth. On the other hand,
"tooth bud" means a tissue at a stage between the transitional stage from
"tooth germ" used in the present invention, that is, the stage where the
accumulation of dentin and enamel characteristic to the hard tissue of a
tooth starts, and the stage before a tooth germinates from gingiva to
exert typical functions of a tooth. Development of a tooth from a tooth
germ follows each of the bud stage, the cap stage, the early bell stage
and the late bell stage. Here, at the bud stage, epithelial cells
invaginate such that they wrap around mesenchymal cells, and when
reaching the early bell stage and the late bell stage, the epithelial
cell portion becomes the outer enamel and the mesenchymal cell portion
begins to form dentin internally. Therefore, a tooth is formed from a
tooth germ by cell-cell interaction between epithelial cells and
mesenchymal cells.

[0054] In the present invention, "mesenchymal cell" means a cell derived
from a mesenchymal tissue and "epithelial cell" means a cell derived from
an epithelial tissue.

[0055] Further, in the present invention, "periodontal tissue" means
alveolar bone and periodontal ligament formed mainly in the outer layer
of a tooth. Alveolar bone and periodontal ligament may be morphologically
and easily identified by those skilled in the art by histological
staining or the like.

[0056] In the reconstructed tooth germ-formation step of the present
invention, a first cell mass formed from either mesenchymal cells or
epithelial cells and a second cell mass formed from the other of the
mesenchymal cells, one of the mesenchymal cells or epithelial cells being
derived from a tooth germ, are positioned in a support carrier, the first
and second cell masses being not mixed with each other but made to
closely contact each other; and then the first and second cell masses are
cultured to form a reconstructed tooth germ.

[0057] Regarding the reconstructed tooth germ-formation step, the method
described in WO 2006/129672 can be applied as it is.

[0058] Each of the first cell mass and the second cell mass is formed from
only mesenchymal cells or epithelial cells, and each of the cell masses
substantially consists of one of these types of cells. The term "cell
mass" means a state wherein cells are closely packed, and the cells may
be either in the state of a tissue or in the state of a cell mass (cell
aggregate) prepared from the state of single cells. The term
"substantially consists" means that the amounts of matters included other
than the cells of interest are as small as possible. The number of cells
constituting each cell mass varies depending on the animal species and on
the type, hardness and size of the support carrier, and may be generally
101 to 108 cells, preferably 103 to 108 cells per
cell mass.

[0059] In terms of the mesenchymal cells and the epithelial cells used in
the present invention, at least one of these may be derived from a tooth
germ so that they may reproduce the cell arrangement in a living body and
effectively form a tooth having a specific structure and directionality,
and, for secure formation of a tooth, both of the mesenchymal cells and
the epithelial cells are most preferably derived from a tooth germ(s).
The tooth germ is preferably at a stage from the bud stage to the cap
stage in view of the fact that the cells are immature and homogeneous in
terms of the stage of differentiation.

[0060] Examples of the mesenchymal cells derived from other than a tooth
germ include cells derived from other mesenchymal tissues in the living
body, such as, preferably, bone marrow cells not containing blood cells
and mesenchymal stem cells, more preferably, mesenchymal cells in the
oral cavity, bone marrow cells inside the jawbone, mesenchymal cells
derived from cranial neural crest cells, mesenchymal precursor cells
which can generate the mesenchymal cells, and stem cells thereof.

[0061] Examples of the epithelial cells derived from other than a tooth
germ include cells derived from other epithelial tissues in the living
body, such as, preferably, epithelial cells of skin, mucosa in the oral
cavity, and gingiva, and more preferred examples of the epithelial cells
include immature epithelial precursor cells which can produce
differentiated, for example, keratinized or parakeratinized, epithelial
cells such as skin, mucosa and the like. Examples of such immature
epithelial precursor cells include non-keratinized epithelial cells and
stem cells thereof.

[0062] The tooth germ and other tissues may be collected from the jawbone
or the like of various animals, for example, primates such as humans and
monkeys and ungulates such as pigs, cows and horses, which are mammals;
rodents such as mice, rats and rabbits, which are small mammals; and dogs
and cats and the like. For the collection of the tooth germ and the
tissue, a condition generally used for collecting a tissue may be applied
without modification, and the tooth germ or the tissue may be collected
under sterile conditions and stored in an appropriate preservation
solution. Examples of a human tooth germ include the tooth germ of a
third molar, which is the so-called wisdom tooth, as well as a fetal
tooth germ, and, from the viewpoint of utilization of autogenous tissues,
usage of the tooth germ of a wisdom tooth is preferred. In the case of
mouse, a tooth germ at an embryonic day of 10 days to 16 days is
preferably used.

[0063] Preparation of mesenchymal cells and epithelial cells from this
tooth germ is carried out by dividing the tooth germ isolated from its
surrounding tissue, into a tooth germ mesenchymal tissue and a tooth germ
epithelial tissue based on their shapes. In this process, enzymes may be
used for the sake of simple isolation of the tissues. Examples of the
enzymes used for such a purpose include dispase, collagenase and trypsin.

[0064] The mesenchymal cells and epithelial cells may be prepared into
single cells from the mesenchymal tissue and the epithelial tissue,
respectively. In order to make the cells of the tissues easily
dispersible into single cells, enzymes such as dispase, collagenase and
trypsin may be used.

[0065] As the medium used for the culture, a medium generally used for
animal cell culture, such as Dulbecco's Modified Eagle Medium (DMEM), may
be used, and serum for promotion of cell proliferation may be added, or,
as an alternative to the serum, a cellular growth factor such as FGF, EGF
or PDGF or a known serum component such as transferrin may be added. In
cases where serum is added, its concentration may be changed
appropriately depending on the culture conditions, and may usually be 10%
by volume. For the cell culture, normal culture conditions, such as those
for culture in an incubator at 37° C. under 5% CO2, may be
applied. An antibiotic such as streptomycin may be added as appropriate.

[0066] The support carrier used in the present invention may be one in
which cells can be cultured, and is preferably a mixture with the above
medium. Examples of such a support carrier include collagen, agarose gel,
carboxymethyl cellulose, gelatin, agar, hydrogel, elastin, fibrin,
fibronectin, laminin, extracellular matrix mixtures, polyglycolic acid
(PGA), polylactic acid (PLA), lactic acid/glycolic acid copolymers
(PLGAs), Cellmatrix (trade name; manufactured by Nitta Gelatin Inc.),
Mebiol Gel (trade name; manufactured by Ikeda Scientific Co., Ltd.) and
Matrigel (trade name; manufactured by Beckton, Dickinson and Company),
which may also be used in combination. These support carriers may have
hardness with which the cells can be virtually maintained at the
locations where they were positioned in the support carrier, and examples
of the support carrier include those in the forms of a gel, fiber and
solid. Among these, from the viewpoint that appropriate hardness and
retentivity can be easily provided, collagen, agarose gel, carboxymethyl
cellulose, gelatin, agar, hydrogel, Cellmatrix, Mebiol Gel, Matrigel,
extracellular matrix mixtures, elastin, fibrin, fibronectin and laminin,
and combinations thereof are more preferred; and collagen, fibrin,
fibronectin, laminin, extracellular matrix mixtures, Cellmatrix, Mebiol
Gel, Matrigel, and combinations thereof are still more preferred. These
support carriers allow achievement of good cell-cell interactions among
the mesenchymal cells and epithelial cells constituting the respective
cell masses and a good interaction between the cell masses. Here, the
hardness with which the cells can be maintained at their locations may be
hardness which is usually applicable to three-dimensional culture, that
is, hardness with which the arrangement of the cells can be maintained
while hypertrophy of the cells due to their growth is not inhibited, and
such hardness can be easily determined.

[0067] Here, the support carrier may have a thickness sufficient for
allowing growth of the first and second cell masses inside the carrier,
and the thickness may be appropriately set depending on the size of the
tissue of interest, and the like.

[0068] Further, the support carrier may have a retentive capacity whereby
the cells can maintain their contacting state without being dispersed. As
used herein, the "contacting state" is preferably a closely-packed (high
density) state which ensures the cell-cell interaction within each cell
mass and between the cell masses, and such a high density state in a cell
aggregate enables culturing of the cells with at a degree of retention,
for example, with which a stronger contacting state than a state of
simply touching can be maintained. For example, in the case of collagen,
appropriate hardness is provided for usage at a final concentration of 2
to 3 mg/ml, that is, a concentration which exerts a jelly strength of 120
g to 250 g based on the method according to J1S-K6503-1996 (measured as
the load necessary for depressing by 4 mm using a plunger with a diameter
of 12.7 mm). The jelly strength is not limited, and other types of
support carriers may also be preferably used as the support carrier of
the present invention as long as these have the same strength based on
the same evaluation method. Further, a support carrier having hardness
corresponding to the desired jelly strength may be obtained by mixing one
or more kinds of support carriers.

[0069] A high density state means a density almost equivalent to the
density at which a tissue is constructed, for example, in the case of the
cell masses, 5×107 to 1×109 cells/ml at the time of
cell positioning, preferably 1×108 to 1×109
cells/ml to ensure the cell-cell interaction without impairing the cell
activity, and most preferably 2×108 to 8×108
cells/ml. In order to prepare a cell mass having such a cell density, it
is preferred to mass and precipitate cells by centrifugation since this
conveniently enables achievement of the high density without impairing
the cell activity. Such centrifugation may be carried out at a revolution
speed equivalent to a centrifugal force of 300 to 1200×g, which
will not adversely affect the survival of the cells, and preferably 500
to 1000×g, for 3 to 10 minutes. Centrifugation at lower than
300×g may lead to insufficient precipitation of the cells and the
cell density may become low, while centrifugation at higher than
1200×g may cause damage to the cells, and therefore both of these
cases are not preferred.

[0070] In cases where high density cells are prepared by centrifugation,
the centrifugation is normally carried out after preparing a suspension
of single cells in a container such as a tube used for cell
centrifugation, and the supernatant is removed to the greatest extent
possible, leaving the cells as the precipitates.

[0071] In cases where the precipitates are prepared by centrifugation,
these may be directly positioned inside the support carrier. Here, the
volume of components other than the cells of interest (for example, a
culture medium, a buffer solution, the support carrier or the like) is
preferably not more than the volume of the cells, and most preferably,
components other than the cells of interest are not contained. In such a
high density cell mass, cells are in close contact with each other and
the cell-cell interaction may be effectively exerted. Especially, in
cases where a cell mass containing only an extremely small amount of
components other than the cells of interest is positioned inside the
support carrier, the cells further aggregate due to solidification of the
support carrier and the like, to provide a state wherein the cells are
more tightly packed.

[0072] The closer the contact between the first cell mass and the second
cell mass, the better, and it is especially preferred that the second
cell mass be positioned such that it presses against the first cell mass.
Further, wrapping around the first cell mass and the second cell mass
with a solid which does not inhibit a culture medium or oxygen permeation
is also effective in making the contact between the cell masses closer.
It is also preferred to add a high-density cell suspension to a solution
having a different viscosity to position the cell suspension therein,
followed by solidification of the solution without any change, since this
may conveniently achieve maintenance of contacting of the cell. Here, in
cases where the first cell mass is an mass of single tooth germ
mesenchymal cells and the second cell mass is a tooth germ epithelial
tissue, it is preferred to position the enamel knot of the tooth germ
epithelial tissue in contact with the first cell mass, but the present
invention is not limited to this.

[0073] When the support carrier is in the form of a gel, solution or the
like, the positioning of the cells in the support carrier may be followed
by a solidification step in which the support carrier is solidified. For
solidification of the support carrier, conditions generally used for
solidification of the support carrier may be applied without
modification. For example, in cases where a solidifiable compound such as
collagen is used for the support carrier, solidification can be achieved
under conditions generally applied, for example, by being left to stand
at the culture temperature for several minutes to several tens of
minutes. By this, adhesions between the cells inside the support carrier
can be fixed and made robust.

[0074] The time period of the culture for formation of a reconstructed
tooth germ varies depending on the number of cells positioned arranged in
the support carrier and the states of the cell masses, the conditions
under which the culture is carried out, as well as on the species of the
animal, and may be generally preferably at least 1 day, more preferably
not less than 3 days. A reconstructed tooth germ or tooth obtained by
culture for this time period can erupt as a functional tooth even after
being embedded in the missing area.

[0075] Further, by extending this culture period, the process of formation
of a reconstructed tooth germ, that is, accumulation of dentin and
enamel, formation of a crown and formation of a root, proceeds.
Therefore, this culture period for formation of a reconstructed tooth
germ can be appropriately controlled depending on the conditions under
which the culture is carried out, the animal species, the condition of
the reconstructed tooth germ, and the like. For example, when the organ
culture described below is carried out, one may proceed to the embedding
step after 7 days, or, in some cases, 6 days, of the culture.
Alternatively, depending on the condition of the reconstructed tooth
germ, or when another culture method is used, the culture period may be
longer than 30 days, or, in some cases, longer than 50 days, longer than
100 days, or longer than 300 days.

[0076] The culture in the support carrier may be carried out either only
with the support carrier containing the first and second cell masses
therein, or in the presence of other animal cells.

[0077] When the culture in the support carrier is carried out only with
the support carrier, it may be carried out under conditions generally
used for culturing of animal cells. For such a culture, in general, the
conditions for culturing of animal cells may be applied without
modification, and the above-mentioned conditions can be applied without
modification. Further, serum derived from mammals, and various cellular
factors which are known to be effective in growth and differentiation of
these cells may be added to the culture. Examples of such cellular
factors include FGF and BMP.

[0078] Further, from the viewpoint of gas exchange and nutrient supply for
tissues and cell masses, and from the viewpoint that the preparation can
be carried out in vitro throughout the whole process without contacting
of the cells with other animal cells, the culture in the support carrier
is preferably carried out as organ culture. In organ culture, generally,
culturing is performed by floating porous membrane on a culture medium
suitable for growth of animal cells and placing the first and second cell
masses embedded in a support carrier on the membrane. The porous membrane
used herein is preferably a membrane having many pores with a diameter of
0.3 to 5 μm, and specific examples thereof include Cell Culture Insert
(trade name) and Isopore Filter (trade name).

[0079] The culture period in organ culture may be about 1 day to 7 days,
and may be preferably 3 to 6 days.

[0080] When the culture in the support carrier is carried out in the
presence of other animal cells, a tooth having a specific cell
arrangement can be formed at an early stage in response to the actions of
various cytokines and the like from the animal cells. Such culture in the
presence of other animal cells may be performed by culture ex vivo using
isolated cells or cultured cells.

[0081] Preferred examples of animals which can be used for this
application include mammals such as humans, pigs and mice, and the
species is preferably the same as that from which the tooth germ tissue
was obtained, or another species which was altered to be immunodeficient.
When a living body is used, preferred examples of transplantation to a
suitable site in the living body include subrenal capsule, mesenteric and
subcutaneous transplantation, which allow an organ or tissue composed of
animal cells to develop as normally as possible. The period of the
culture in such presence of animal cells may be appropriately controlled
depending on the origin of the cells, conditions of the culture, the
animal species to which the transplantation is carried out, and the like,
as in the case of the organ culture described above.

[0082] By such a reconstructed tooth germ-formation step, a tooth germ
which may have a cell arrangement (structure) specific to a tooth, that
is, dentin inside and enamel outside, or a tooth having these, can be
obtained. Further, the tooth germ or the tooth also preferably has
directionality, that is, a tip (crown) and a root of a tooth.

[0083] Both the first cell mass and the second cell mass are preferably
masses of single cells since an aggregate of teeth composed of plural
teeth having a cell arrangement specific to a tooth can be obtained. When
such an aggregate of teeth was obtained, each tooth is separated from the
aggregate before its use.

[0084] In the directionality-confirmation step in the method of production
of a restorative material of the present invention, the directionality of
the tooth germ or tooth obtained in the reconstructed tooth
germ-formation step described above is confirmed so as to enable the
tooth germ or tooth to be embedded in the tooth-deficient area such that
the tip of the tooth faces the interior of the oral cavity.

[0085] The directionality of the tooth germ or tooth means the
directionality with which the tooth germ or tooth is embedded in the
tooth-deficient area. When formation of a crown is found, the crown is
confirmed to be facing the interior of the oral cavity, and when
formation of a crown cannot be found, the epithelial cell layer in the
portion corresponding to the crown or the epithelial cell layer in the
reconstructed tooth germ is confirmed to be facing the interior of the
oral cavity. Alternatively, the open portion of the
epithelial/mesenchymal cell layer of the reconstructed tooth germ may be
confirmed to be at the opposite side with respect to the oral cavity. By
positioning the tooth germ or tooth in the tooth-deficient area according
to the directionality confirmed as above, the tip (crown) of the tooth
can be made to face the interior of the oral cavity, so that the
direction of the tooth becomes the same as that of the surrounding teeth.

[0086] The tooth germ or tooth whose directionality has been confirmed in
this directionality-confirmation step is used as a restorative material
to obtain an equivalent of the missing tooth in the tooth-deficient area.

[0087] That is, the restorative material containing the tooth germ or
tooth after confirmation of its directionality is embedded in the
tooth-deficient area. The tooth germ or tooth in the restorative material
grows thereafter in the missing area over time and erupts as an
equivalent of a tooth. The tip (cusp) of the erupted tooth equivalent
reaches almost the same position as those of the surrounding teeth
(occlusal plane), and the tooth equivalent does not extend further
thereafter, although the situation varies depending on the state, size
and the like of the tooth germ or tooth in the restorative material. The
length of the period during which the tooth germ or tooth remains
embedded until eruption and occlusion is different among animal species
and varies depending on the condition of the embedded tooth germ or
tooth.

[0088] The erupted tooth equivalent has hardness similar to that of a
native tooth. The hardness is an index that indicates the extent to which
the dentin and the enamel of the tooth equivalent tolerate deformation
due to compression, abrasion and/or the like, and can be confirmed by
measurement of the Knoop hardness. The Knoop hardness of enamel of a
normal tooth in an adult mouse is 300 to 600 KHN, preferably 300 to 500
KHN, and the Knoop hardness of normal dentin is 60 to 120 KHN. The tooth
equivalent obtained in the present invention has Knoop hardness within
these ranges. By this, masticatory function equivalent to that of a
normal tooth is exerted.

[0089] Further, the erupted tooth equivalent has dental pulp and
periodontal ligament in addition to enamel and dentin, showing a tissue
constitution similar to that of a normal tooth. Since the tooth has the
periodontal ligament, nerves extend, so that responsiveness to noxious
stimuli such as compression to the tooth is maintained. Such a tissue
constitution can be confirmed visually or by histological analysis,
immunostaining, analysis of gene expression, or the like according to a
conventional method.

[0090] Thus, the restorative material obtained by the method of the
present invention for producing a restorative material can be used as a
raw material for a tooth equivalent having a length and hardness which
allow its occlusion with the opposing tooth.

[0091] In the embedding step in the method of the present invention for
restoring a tooth-deficient area, a tooth germ or tooth obtained by the
reconstructed tooth germ-formation step described above is embedded in a
tooth-deficient area as a restorative material.

[0092] Here, in terms of the direction in which the tooth germ or tooth is
embedded, the tooth germ or tooth is preferably positioned in the same
manner as in the directionality-confirmation step in the above-mentioned
method for production of a restorative material, such that the crown
faces the interior of the oral cavity when formation of a crown is found,
or such that the epithelial cell layer in the portion corresponding to
the crown or the epithelial cell layer in the reconstructed tooth germ
faces the interior of the oral cavity when formation of a crown cannot be
found, or alternatively, such that the open portion of the
epithelial/mesenchymal cell layer of the reconstructed tooth germ is
positioned at the opposite side with respect to the oral cavity. By this,
the tip (crown) of the tooth equivalent can be made to face the interior
of the oral cavity, so that the direction of the tooth equivalent becomes
the same as that of the surrounding teeth.

[0093] The size and the depth of the missing area are usually not
restricted as long as the area is formed in gingiva caused by extraction
of a tooth, or the like, and the shape of the missing area is not
restricted. As long as a regenerated tooth germ can be embedded in the
area, these can be appropriately controlled depending on the missing
place, subject animal species, type of the tooth of interest, and the
like.

[0094] Such a missing area is usually positioned on the jawbone, alveolar
bone of the oral cavity, or the like. When the mass of the alveolar bone
is decreased due to loss of a tooth, the bone mass may be increased by
bone regeneration according to a known method clinically used for
placement of an implant, such as the GTR (guided tissue regeneration)
method. The positioning of the tooth germ or tooth in the hole portion is
preferably followed by suture or the like according to a conventional
process.

[0095] Thereafter, the embedded tooth germ grows with time to become a
tooth in the missing area, followed by its eruption as a tooth
equivalent. Similarly to the matters described above for the method for
production of a restorative material, the tip (cusp) of the erupted tooth
equivalent reaches almost the same position as those of the surrounding
teeth (occlusal plane), and the tooth equivalent does not extend further
thereafter, although the situation varies depending on the state, size
and the like of the tooth germ to be embedded. By this, the length of the
tooth can be made to allow occlusion of the tooth with the opposing
tooth. The length of the period during which the tooth germ or tooth
remains embedded until eruption and occlusion is different among animal
species and varies depending on the condition of the embedded tooth germ
or tooth.

[0096] Similarly to the fact described above for the method for production
of a restorative material, the erupted tooth equivalent has hardness
similar to that of a native tooth. The hardness is an index that
indicates the extent to which the dentin and the enamel of the tooth
equivalent tolerate deformation due to compression, abrasion and/or the
like, and can be confirmed by measurement of the Knoop hardness. The
Knoop hardness of enamel of a normal tooth in an adult mouse is 300 to
600 KHN, preferably 300 to 500 KHN, and the Knoop hardness of normal
dentin is 60 to 120 KHN. The tooth equivalent obtained in the present
invention has Knoop hardness within these ranges. By this, masticatory
function equivalent to that of a normal tooth is exerted.

[0097] Further, similarly to the fact described above for the method for
production of a restorative material, the erupted tooth equivalent has
dental pulp and periodontal ligament in addition to enamel and dentin,
showing a tissue constitution similar to that of a normal tooth. Since
the tooth has the periodontal ligament, nerves extend, so that
responsiveness to noxious stimuli such as compression to the tooth is
maintained. Such a tissue constitution can be confirmed visually or by
histological analysis, immunostaining, analysis of gene expression, or
the like according to a conventional method.

[0098] In the method of the present invention for restoring an area
missing a tooth, a tooth-deficient area is restored using a tooth germ or
tooth reconstructed as described above, and therefore the tooth-deficient
area can be restored such that normal occlusion, and responsiveness to
noxious stimuli equivalent to that of a normal tooth, are attained. As a
result, the tooth equivalent regenerated in the missing area acts as a
functional tooth, and the restored state can be maintained for a long
period of time.

[0099] This enables maintenance of quality of life, and therefore the
present method is suitably used in the field of esthetics. Further, since
deterioration of health conditions due to the existence of a
tooth-deficient area in the oral cavity can be avoided, the health
conditions of non-human mammals such as livestock including cows, horses
and pigs and pet animals including dogs and cats can be maintained.

EXAMPLES

[0100] Examples of the present invention are now described, but the
present invention is not limited thereto. "%" in Examples is by weight
(mass) unless otherwise specified.

[0101] In order to form a tooth, a tooth germ was reconstructed. Mouse was
used as a model of this experiment.

[0102] A tooth germ tissue of a mandibular molar was isolated from an
embryo of a C57BL/6N mouse (purchased from CLEA Japan, Inc.) at the
embryonic day of 14.5 days under the microscope according to a
conventional method. The tooth germ tissue of a mandibular molar was
washed with Ca2+/Mg2+-free phosphate buffer (PBS(-)), and
treated at room temperature for 12.5 minutes with an enzyme solution
prepared by adding Dispase II (Roche, Mannheim, Germany) to PBS(-) to a
final concentration of 1.2 U/ml. The tooth germ tissue was then washed 3
times with DMEM (Sigma, St. Louis, Mo.) supplemented with 10% FCS (JRH
Biosciences, Lenexa, Kans.). Further, a DNase I solution (Takara, Siga,
Japan) was added to a final concentration of 70 U/ml to disperse the
tooth germ tissue, and the tooth germ epithelial tissue and the tooth
germ mesenchymal tissue from each other were separated using a 25 G
injection needle (Terumo, Tokyo, Japan).

[0103] In terms of tooth germ epithelial cells, the tooth germ epithelial
tissue obtained as described above was washed 3 times with PBS(-), and
subjected to 2 times of treatment at 37° C. for 20 minutes with an
enzyme solution which Collagenase I (Worthington, Lakewood, N.J.) was
dissolved at a final concentration of 100 U/ml in PBS(-). The cells were
collected by precipitation by centrifugation, and treated with 0.25%
Trypsin (Sigma)-PBS(-) at 37° C. for 5 minutes. The cells were
washed 3 times with DMEM supplemented with 10% FCS, and a DNase I
solution was added to the cells to a final concentration of 70 U/ml,
followed by pipetting to obtain single tooth germ epithelial cells.

[0104] On the other hand, in terms of tooth germ mesenchymal cells, the
tooth germ mesenchymal tissue was washed 3 times with PBS(-), and
subjected to treatment with PBS(-) containing 0.25% Trypsin (Sigma) and
50 U/ml Collagenase I (Worthington). DNase I (Takara) was added to the
resultant to 70 U/mI, followed by pipetting to obtain single tooth germ
mesenchymal cells.

(2) Preparation of Reconstructed Tooth Germ

[0105] Subsequently, using the tooth germ epithelial cells and the tooth
germ mesenchymal cells prepared as described above, a tooth germ was
reconstructed. In a 1.5 mL microtube (Eppendorf, Hamburg, Germany) to
which silicone grease was applied, the tooth germ epithelial cells or the
tooth germ mesenchymal cells suspended in DMEM (Sigma) supplemented with
10% FCS (JRH) were placed, and the cells were recovered by centrifugation
as a precipitate. The supernatant of the culture medium after the
centrifugation was removed as much as possible, and centrifugation was
carried out again, followed by complete removal of the culture medium
remaining around the precipitate of the cells under a stereoscopic
microscope using GELoader Tip 0.5-20 μL (Eppendorf). Thereafter, the
cells were dispersed by tapping or pipetting, to prepare cells to be used
for preparation of a reconstructed tooth germ.

[0106] To a petri dish to which silicone grease was applied, 30 μL of
Cellmatrix type I-A (Nitta gelatin, Osaka, Japan) was added dropwise to
prepare a collagen gel droplet. To this solution, 0.2 to 0.3 μL of the
above tooth germ epithelial cells or tooth germ mesenchymal cells were
applied using a 0.1-10 μL pipette tip (Quality Scientific plastics) to
prepare a cell aggregate (5×104 cells/aggregate). In terms of
a reconstructed tooth germ, tooth germ epithelial cells were applied in
the same manner such that these cells were brought into contact with the
cell aggregate of tooth germ mesenchymal cells prepared above, to prepare
a cell aggregate, thereby preparing a reconstructed tooth germ wherein
the both are in close contact with each other.

[0107] The reconstructed tooth germ prepared in the gel was left to stand
in a CO2 incubator for 10 minutes to solidify Cellmatrix type I-A
(Nitta Gelatin). A culture vessel was prepared such that DMEM (Sigma)
supplemented with 10% FCS (JRH) was in contact with Cell Culture Inserts
(PET membrane having a pore size of 0.4 μm; BD, Franklin Lakes, N.J.).
Onto the membrane of Cell Culture Inserts in the culture vessel, the cell
aggregate was transferred together with the surrounding gel as a support
carrier, followed by organ culture for 18 to 24 hours. After the culture,
organ culture was continued in the Cell Culture Inserts to analyze
development of a tooth.

(3) Method of Individual Separation

[0108] On Day 2 to 5 during the organ culture, a reconstructed tooth germ
in which plural tooth germs were developing was subjected to surgical
separation into individual tooth germs under a stereoscopic microscope
using an injection needle and forceps. To a petri dish to which silicone
grease was applied, 30 μL of Cellmatrix type I-A (Nitta gelatin,
Osaka, Japan) was added dropwise to prepare a collagen gel droplet. Each
individually separated tooth germ was placed in this solution, and left
to stand in a CO2 incubator for 10 minutes to solidify Cellmatrix
type I-A (Nitta Gelatin). A culture vessel was prepared such that DMEM
(Sigma) supplemented with 10% FCS (JRH) was in contact with Cell Culture
Inserts (PET membrane having a pore size of 0.4 μm; BD, Franklin
Lakes, N.J.). Onto the membrane of Cell Culture Inserts in the culture
vessel, the individually separated tooth germ was transferred together
with the surrounding gel as a support carrier, followed by organ culture
for 18 to 24 hours.

(4) Intraoral Transplantation

[0109] From the individually separated tooth germ prepared as described
above, the surrounding gel was surgically removed using an injection
needle and forceps. The individually separated tooth germ was
transplanted to alveolar bone in the first molar (M1) region of the upper
jaw of C57BL/6 of 7 to 10 weeks old, without being transplanted to
subrenal capsule. By the present invention, an individual tooth was
allowed by erupt in the oral cavity, to realize oral function such as
mastication.

Example 2

(1) Preparation of Individually Separated Tooth Germ

[0110] Organ culture was carried out in the same manner as in Example 1(1)
to (2), and on Day 2 to 5 during the organ culture, a reconstructed tooth
germ in which plural tooth germs were developing was subjected to
surgical separation into individual tooth germs under a stereoscopic
microscope using an injection needle and forceps. In terms of the
individually separated tooth germ, to a petri dish to which silicone
grease was applied, 30 μL of Cellmatrix type I-A (Nitta gelatin,
Osaka, Japan) was added dropwise to prepare a collagen gel droplet. Each
individually separated tooth germ was placed in this solution, and left
to stand in a CO2 incubator for 10 minutes to solidify Cellmatrix
type I-A (Nitta Gelatin). A culture vessel was prepared such that DMEM
(Sigma) supplemented with 10% FCS (JRH) was in contact with Cell Culture
Inserts (PET membrane having a pore size of 0.4 μm; BD, Franklin
Lakes, N.J.). Onto the membrane of Cell Culture Inserts in the culture
vessel, the individually separated tooth germ was transferred together
with the surrounding gel as a support carrier, followed by organ culture
for 5 days. When plural reconstructed tooth germs were formed, these were
surgically separated into single reconstructed tooth germs. After the
culture, the surrounding gel was surgically removed using an injection
needle and forceps, and the tooth germ was transplanted to alveolar bone
in the first molar (M1) region of the upper jaw of C57BL/6 of 8 weeks
old.

(2) Method of Intraoral Transplantation

[0111] Three days before intraoral transplantation, 240 μl/20 g body
weight of physiological saline containing 5 mg/ml sodium pentobarbital
was intraperitoneally injected to an 8-week old C57BL/6 which had been
subjected to inhalation anesthesia with diethyl ether. An upper jaw M1 of
the mouse under anesthesia was extracted from the upper jaw using
forceps, and absence of a residual root was confirmed, followed by wiping
spouting blood with absorbent cotton to stop bleeding. In terms of food
ingestion, the mouse was fed with powdered diet every day. The mouse was
kept for not less than 3 weeks to allow curing of the extraction wound,
to prepare C57BL/6 lacking M1.

[0112] The C57BL/6 lacking M1, prepared by the method described above, was
subjected to inhalation anesthesia with diethyl ether, and 240 μl/20 g
body weight of physiological saline containing 5 mg/ml sodium
pentobarbital was intraperitoneally injected to the mouse. The mouse
under anesthesia was fixed on its back on a dissecting table, and the
upper and lower jaws were fixed using rubbers or threads such that the
mouth was fully open. The gingiva at the alveolar bone crest in the upper
jaw M1 region was incised using a scalpel, and peeled off together with
periosteum to expose the mandible. Using a pin vise (extra fine drill), a
hole having a diameter of 1 mm was prepared in the region of the mandible
corresponding to M1 such that perforation of the maxillary sinus was not
caused, and the individually separated tooth germ was transplanted to the
hole. After the transplantation, the wound was closed with the
periosteum/gingival flap, and sutured with thread with an attached
needle. In order to control the direction of the individually separated
tooth germ to be transplanted, the cusp of the individually separated
tooth germ was labeled with methylene blue, and the transplantation was
carried out such that the label was facing the direction of eruption. The
8-week old C57BL/6 subjected to the intraoral transplantation was fed
with powdered diet every day

[0113] To the C57BL/6 to which an individual tooth germ was transplanted
by the method described above, 240 μl/20 g body weight of
physiological saline containing 5 mg/ml sodium pentobarbital was
intraperitoneally injected. The mouse under anesthesia was fixed on its
back on a dissecting table, and the upper and lower jaws were fixed using
rubbers such that the mouth was fully open. The regenerated tooth, the
distal areas of M2 and M3, and the surrounding gingiva were observed
before eruption, immediately before eruption, immediately after eruption,
during eruption, and when the tooth reached the occlusal surface. The
observation results are shown in FIG. 1. In each of FIG. 1A to FIG. 1E,
the left panel shows the result obtained during the eruption period
(before eruption to immediately after eruption); the middle panel shows
the result obtained during the period after eruption to occlusion; and
the right panel shows the result obtained during the period after
occlusion. In each panel of each of FIG. 1A to FIG. 1E, the regenerated
tooth is shown on the right side of the panel.

[0114] About 20 to 50 days after the transplantation, the cusp of the
regenerated tooth could be observed at the M1 gingiva region, indicating
that eruption has started. The regenerated tooth increased its crown
height over time and reached the occlusal plane. The regenerated tooth
which reached the occlusal plane attained intercuspation with the
opposing tooth (see FIG. 1A to FIG. 1C).

[0115] From these results, it was revealed that the regenerated tooth
forms a stable occluding relation similarly to a normal tooth and has a
masticatory function.

(3) MicroCT Analysis

[0116] The head containing the maxilla to which the individually separated
tooth germ was transplanted was removed, and the whole head was fixed in
4% paraformaldehyde-phosphate buffer for 12 hours, followed by CT imaging
using inspeXio SMX-90CT (manufactured by Shimadzu Corporation). The CT
imaging was carried out with the settings of: view number, 600; average,
10; scan, 1; image, 512×512; scaling, 50; slice thickness, 1; and
without BHC. The data obtained by the CT imaging were 3D-reconstructed
using an analysis software Imaris (manufactured by Zeiss), to prepare
cross-sectional images of MicroCT.

[0117] As a result of the MicroCT analysis, it was revealed, as shown in
FIG. 1D and FIG. 1E, that, in the eruption process, the regenerated tooth
longitudinally moves while forming its root, and erupts from the alveolar
bone crest, after which the tooth reaches the occlusal plane. A void
corresponding to the periodontal ligament could be observed between the
root portion and the alveolar bone, and, in the cross-sectional view of
the tooth, the pulp cavity existed and an apical foramen opens in the
root apex, similar to a normal tooth. It was revealed that the
regenerated tooth attained intercuspation with the opposing tooth in the
occluded state. From these results, it was revealed that, during the
eruption process, the regenerated tooth longitudinally moves in the
alveolar bone with retaining the periodontal ligament and the pulp
cavity, and reaches the occlusal plane, similarly to a normal tooth.

[0118] Further, similarly, M3 in the upper and lower jaws in the head
fixed in 4% paraformaldehyde-phosphate buffer were exposed, and intraoral
images at the centric occlusion position was taken according to a
conventional method wherein the horizontal angle was determined based on
the plane between the occlusal surfaces of the mandibular M3 and the
mandibular M1, and the vertical angle was determined based on the overlap
between the mesial buccal cusp and the mesial lingual cusp of the
mandibular M1 seen from the lateral direction.

[0119] As a result, it was confirmed that the regenerated tooth was
occluding with the distal cusp of the mandibular M1, establishing a flat
occlusal plane together with the maxillary M2 and M3. From these results,
it was suggested that the erupted regenerated tooth has an established
occlusal plane which is appropriate for induction of the mandibular
position.

(4) Knoop Hardness

[0120] An individual tooth which was allowed to develop in the oral cavity
in Example 2(1) was recovered, and the hardness of each of enamel and
dentin of the tooth was measured for the identical tooth at not less than
3 positions.

[0121] The measurement was carried out using a microhardness tester
(HM-102, manufactured by Mitutoyo Corporation) equipped with a
quadrangular pyramidal diamond indenter for measurement of Knoop Hardness
having two vertically opposite angles 172°30' and 130°
(19BAA061, manufactured by Mitutoyo Corporation). The indenter was
pressed into the regenerated tooth with a load of 10 g for 10 seconds,
and the hardness was calculated from the length of the longer side of the
7.11:1 rhombic impression. The regenerated tooth was fixed on a metal
plate using a dental resin (Unifast III, manufactured by GC CORPORATION).
Measurement for enamel was carried out for a portion parallel to the
ground, and measurement for dentin was carried out by cutting the tooth
in the direction horizontal to the ground using a dental mechanical
engine (ULTIMATE 500, manufactured by NAKANISHI INC.) to expose the
surface to be tested. The results of the measurement of the Knoop
hardness are shown in FIG. 2.

[0122] As shown in FIG. 2, the average Knoop hardness of enamel of a
normal tooth was 341.083 KHN at 3 weeks old, 457.5 KHN at 6 weeks old,
and 436 KHN at 9 weeks old; and the average Knoop hardness of enamel of
the regenerated tooth was 469.81 KHN (see FIG. 2A). The average Knoop
hardness of dentin of a normal tooth was 66.87 KHN at 3 weeks old, 76.53
KHN at 6 weeks old, and 88.58 KHN at 9 weeks old; and the average Knoop
hardness of dentin of the regenerated tooth was 81.83 KHN (see FIG. 2B).
Thus, the regenerated tooth was suggested to have hardness suitable for
exerting normal masticatory function.

(5) Histological Analysis

[0123] About 20 to 50 days after the transplantation, the cusp of the
regenerated tooth could be observed at the M1 gingiva region, indicating
that eruption has started. The state of eruption was observed, and the
maxilla before eruption, the maxilla immediately before eruption, the
maxilla immediately after eruption, the maxilla during eruption, and the
maxilla when the tooth reached the occlusal surface were removed. The
maxillae were fixed in 4% paraformaldehyde-phosphate buffer for 16 hours
and decalcified in 22.5% formic acid for 72 hours, followed by paraffin
embedding according to a conventional method and preparing 10-μm
sections. The amount of the decalcifying fluid was 50 ml per two
maxillae, and the total volume was replaced at 48 hours during the
decalcification. Histological analysis was carried out by
hematoxylin-eosin staining according to a conventional method.

[0124] FIG. 3 shows the regenerated tooth immediately before eruption
(FIG. 3, white arrow). As shown in FIG. 3, a tissue structure equivalent
to that of a normal tooth, having enamel, dentin, the dental pulp and the
periodontal ligament could be observed. In terms of the respective
tissues, the enamel had enamel prisms radially extending side by side,
and the dentin was found to have dentinal tubules. The periodontal
ligament had a sufficient thickness, and had a structure with which the
masticatory force can be buffered. During the eruption process, it was
observed that the regenerated tooth longitudinally moved while forming
its root, and erupted from the alveolar bone crest, subsequently reaching
the occlusal plane. During this process, the periodontal ligament was
retained, suggesting that the regenerated tooth is in harmony with the
periodontal tissue.

(6) Analysis of Remodeling of Bone by Orthodontic Force

[0125] Since a tooth is bound to the surrounding alveolar bone via the
periodontal ligament, application of orthodontic force to a tooth causes
transmission of the mechanical stress to the surrounding environment via
the periodontal ligament. In the area of the periodontal ligament
compressed by the orthodontic force, absorption of the alveolar bone by
osteoclasts occurs, and, in the area of the periodontal ligament which
has undergone traction, osteogenesis by osteoblasts occurs, thereby
keeping the spatial distance between the tooth and the alveolar bone
constant. For the purpose of evaluation of such functions of the
periodontal ligament, orthodontic force was applied to the regenerated
tooth and remodeling of the bone, such as emergence of osteoclasts in the
compressed side and emergence of osteoblasts in the side which has
undergone traction, caused by movement of a normal tooth, were confirmed
by the following analysis.

[0126] Using nickel-titanium wires having a diameter of 0.012 inch (VIM-NT
orthodontic wire, round type, OralCare), orthodontic wires for buccal
movement were formed such that one end of each wire was bent to form a
loop which can be adapted and fitted to the neck of either incisor in the
maxilla. To C57BL/6 to which an individual tooth germ was transplanted by
the method described above, 240 μl/20 g body weight of physiological
saline containing 5 mg/ml sodium pentobarbital was intraperitoneally
injected. The mouse under anesthesia was fixed on its back on a
dissecting table, and the upper and lower jaws were fixed using rubbers
or threads such that the mouth was fully open. The length of the end of
the wire opposite to the loop was adjusted such that the end reaches the
distal area of the erupted tooth. The loop of the orthodontic wire for
buccal movement was fitted to the neck of either incisor in the maxilla,
while adapting the other end of the wire to the buccal-side neck of the
tooth to be subjected to orthodontics, which loop was then bonded and
fixed with UNIFIL FLOW (photopolymerizable resin, manufactured by GC
CORPORATION). The end of the wire which had been adapted to the
buccal-side neck of the tooth was moved to the lingual-side neck of the
tooth to activate orthodontic force. Subsequently, histological analysis
to analyze the bone remodeling phenomenon caused by the periodontal
ligament was carried out on Day 6 after the orthodontics when the bone
remodeling phenomenon is especially remarkable. The results are shown in
FIG. 4A to FIG. 4C.

[0127] To analyze the responsive function of the periodontal ligament of
the regenerated tooth against mechanical stress, orthodontic force to
cause movement to the buccal side was applied to the regenerated tooth,
and the tissue image of remodeling of the bone was observed with
hematoxylin-eosin-stained images.

[0128] As a result, in the buccal side, compression of the periodontal
ligament, wherein periodontal ligament fibers are compressed due to
narrowing of the periodontal space, was observed (see the black-framed
area B in FIG. 4A, and FIG. 4B). On the other hand, in the opposite side,
that is, the lingual side, the periodontal space expanded, causing
traction of the periodontal ligament, wherein periodontal ligament fibers
became tense (see the black-framed area C in FIG. 4A, and FIG. 4C).
Further, since a single layer of cells aligned along the alveolar bone
wall in the tension side of the periodontal ligament was observed,
emergence of osteoblasts which form the bone (see arrowheads in FIG. 4C)
was suggested. Further, in the pressure side, which is opposite to the
tension side, multinucleated giant cells having a number of nuclei in the
cell body were observed in the alveolar bone, and cavities formed by bone
resorption were observed, so that expression of osteoclasts (see arrows
in FIG. 4B) and bone resorption were suggested.

[0129] From these results, it was suggested that, in the regenerated
tooth, mechanical stress such as orthodontic force causes response of the
periodontal ligament, inducing the bone remodeling phenomenon, as in a
normal tooth.

(8) Analysis of Pain Stimulation

[0130] Pain due to compression of teeth is well-known to be caused by
orthodontics and the like, and it has been revealed that the pain is
mediated by nerves existing especially in the periodontal ligament, and
that production of the c-Fos protein increases in nerve cells in
trigeminal spinal subnucleus caudalis (the medullary dorsal horn) (Byers
M R. et al., Crit Rev Oral Biol Med 10, 4-39, 1999; Deguchi T. et al. J
Dent Res 82:677-681, 2003; Fujiyoshi, Y. et al., Neuroscience Letters
283, 205-208, 2000). Further, pain due to exposure of dental pulp is
well-known to be caused by the dental pulp exposure treatment, and it has
been revealed, as in the case of the pain due to orthodontics, that the
pain is mediated by nerves existing especially in dental pulp, and that
production of the c-Fos protein increases in nerve cells in trigeminal
spinal subnucleus caudalis (the medullary dorsal horn) (Byers M R. et
al., Crit Rev Oral Biol Med 10, 4-39, 1999; Deguchi T. et al. 3 Dent Res
82:677-681, 2003). Thus, in order to analyze nerve function of the
regenerated tooth, the regenerated tooth of the mouse was subjected to
compressive stimulation similar to that applied during orthodontics, and
the dental pulp exposure treatment by drilling a hole in dentin with a
dental drill, and whether or not expression of the c-Fos protein
increases in trigeminal spinal subnucleus caudalis (the medullary dorsal
horn) was analyzed.

[0131] To C57BL/6 to which an individual tooth germ was transplanted by
the method described above, 200 μl/20 g body weight of physiological
saline containing 5 mg/ml sodium pentobarbital was intraperitoneally
injected. The mouse under anesthesia was fixed on its back on a
dissecting table.

[0132] Two hours and 48 hours after the activation of orthodontic force by
the above method, and 2 hours after the dental pulp exposure treatment,
the C57BL/6 was subjected to perfusion fixation in 4%
paraformaldehyde-phosphate buffer. The isolated medulla oblongata was
fixed in 4% paraformaldehyde-phosphate buffer for 16 hours, and embedded
in OCT compound (Miles Inc., Naperville, Ill.) according to a
conventional method, followed by preparing 50-μm sections using a
cryostat (Leica, Wetzlar, Germany). The prepared sections were subjected
to immunostaining using c-fos (SANTA CRUZ BIOTECHNOLOGY, INC., Santa
Cruz, Calif., USA) as a primary antibody, Goat IgG fraction to rabbit IgG
(CAPPEL, Aurora, Ohio, USA) as a secondary antibody, and Rabbit
peroxidase anti-Peroxidase (CAPPEL, Aurora, Ohio, USA) as a tertiary
antibody, and the expression in trigeminal spinal nucleus in the medulla
was compared.

[0133] After removal of OCT compound, the sample was incubated in 0.3%
hydrogen peroxide solution-80% methanol at room temperature for 1 hour to
block endogenous enzyme activities. Subsequently, blocking was carried
out with a blocking solution (3% Goat Serum-TBS) at room temperature for
1 hour, and the primary antibody was allowed to react with the sample at
4° C. for 72 hours. After washing the sample, blocking was carried
out with the blocking solution at room temperature for 1 hour, and the
secondary antibody was allowed to react with the sample at room
temperature for 1 hour. After washing the sample, blocking was carried
out with the blocking solution at room temperature for 1 hour, and the
tertiary antibody was allowed to react with the sample at room
temperature for 1 hour. After sufficient washing, a DAB/NAS substrate
solution (0.08 to 0.1% nickel ammonium sulfate containing 0.04%
DAB-0.003% hydrogen peroxide-TBS) was added dropwise to the sample to
allow coloring. The sections after the coloring were sufficiently washed
and embedded in glycerol. The sample was observed with an upright
microscope (Axioimager, manufactured by Zeiss). The results are shown in
FIG. 5.

[0134] As a result, as shown in FIG. 5A, in trigeminal spinal subnucleus
caudalis (the medullary dorsal horn) in C57BL/6 that was subjected to
neither orthodontics of the regenerated tooth nor the dental pulp
exposure treatment, expression of the c-Fos protein was not observed,
but, as shown in FIG. 5B and FIG. 5C, in trigeminal spinal subnucleus
caudalis (the medullary dorsal horn) in C57BL/6 that was subjected to
orthodontics, a high level of expression of the c-Fos protein was induced
2 hours later, and the expression was maintained until 48 hours after the
stimulation as shown in FIG. 5C. Further, as shown in FIG. 5D, in
trigeminal spinal subnucleus caudalis (the medullary dorsal horn) in
C57BL/6 that was subjected to the dental pulp exposure treatment of the
regenerated tooth, a high level of expression of the c-Fos protein was
induced 2 hours later. From these results, it was suggested that nerve
fibers existing in the periodontal ligament region sense the compressive
stimulation caused upon application of compressive stimulation by
orthodontics to the regenerated tooth, and pain stimulation, and that the
stimulation is transmitted to the central nervous system as in the case
of a normal tooth.

[0135] Thus, it was revealed that, by using the restoration method of the
present invention, a tooth-deficient area can be restored such that the
regenerated tooth has hardness equivalent to a normal tooth, an ability
to achieve normal occlusion, and responsiveness to stimuli equivalent.

[0136] The disclosure of Japanese Patent Application No. 2008-211870 is
hereby incorporated by reference in its entirety.

[0137] All the literature, patent applications and technical standards
described in the present specification are hereby incorporated by
reference to the same extent as in cases where each literature, patent
application or technical standard is concretely and individually
described to be incorporated by reference.